The overall purpose of this project is to gain a better understanding of the mechanisms underlying neurotransmitter release. Its modulation is thought to play an important role in higher order mental process such as learning and memory. Studies in vivo or in slices have allowed limited advances in the analysis of synaptic release properties, because of the lack of direct access to the presynaptic terminal. Cultured systems have real advantages over intact animals and slices, including the possibility of delivering drugs intracellularly to either side of the synapse including the presynaptic terminal, and the possibility of long-term access to cells under controlled environment for genetic manipulation. Thus, cultured hippocampal neurons will be used in our experiments. Our researches will focus on cGMP-dependent protein kinases (cGKs), a family of proteins with a poorly understood function, and their action on transmitter release. The first specific aim will examine whether presynaptic cGK type I and II are involved in basal transmitter release and plasticity. The goal of the second specific aim will be to test whether VASP and CAMKII, two cGK substrates, are cGK targets during neurotransmission and plasticity. cGK involvement in the release machinery and vesicle cycle kinetics will be examined in the third specific aim in order to explore the functional role of the kinase. Finally, cGK regulation of the expression of the presynaptic proteins, synaptophysin and a-synuclein, will be examined by combining immunocytochemical techniques with electrophysiological recordings. Evidence for a mechanistic link between cGKs, increase in the number of synaptophysin and a-synuclein immunoreactive clusters and long-lasting increase in neurotransmitter release will be provided. A better understanding of these mechanisms will yield new drug therapies with potential in treatment of Alzheimer's Disease and other neurodegenerative disorders with staggering social, economic and personal costs to the sufferers, their families and all of society.